During directed neurite extension, growth cones are guided by extrinsic sues, such as cells and extracellular matrix molecules. Recent evidence suggests that microtubule (MT) alignment is important for growth cone branching and turning in response to guidance cues, and that selective MT invasion of growth cone branches may determine the direction of motility. Also, actin filaments may regulate the above reorganization of MTs. However, the role of stable Mts in growth cone branching and turning behaviors is currently unknown. To test the hypothesis that cone branching and turning behaviors depend on temporal and spatial relationships of stable and dynamic MTs relative to actin filaments and substratum contacts, the Specific Aims of this proposal are: 1) To elucidate the temporal and spatial relationships of stable and dynamic MTs relative to the actin filament cytoskeleton and substratum contacts in growth cones branching and turning on various in vitro substrata, 2) To directly probe the dynamic changes in MT orientation and stability by observing fluorescent MTs in living growth cones that are branching and turning. Chick sensory growth cones will be recorded by time-lapse phase contrast and interference reflection video microscopy as they branch on laminin, fibronectin and L1 substrata, and turn at a chondroitin sulfate proteoglycan border. Following immunocytochemical staining for stable and dynamic MTs, actin filaments and focal contact proteins, confocal optical sections and rendered three-dimensional reconstructions will show the organization of stable and dynamic MTs relative to actin filaments, and focal contact proteins relative to known regions of substratum apposition. Microinjection of fluorescent tubulin and subsequent time-lapse fluorescence videomicroscopy will show MT dynamics in living growth cones that are branching and turning. The orientation of recorded MTs will be compared to the arrangement of immunocytochemically stained stable and dynamic MTs by confocal microscopy and three-dimensional reconstructing/rendering. Results of these experiments will indicate the arrangement of stable and dynamic MTs with respect to new branches and the direction of turning, and provide clues about the mechanism of MT advance into new branches, the influence of actin filaments on this MT arrangement, and how these relationships may differ in response to substratum-dependant cues. This project studies the basic mechanisms of axonal growth. Results of these studies will help to better understand the development and regeneration of the nervous system. Ultimately, this work may be critical in prevention and/or treatment of birth defects that involve the nervous system, or in the promotion of nerve regeneration in individuals who have suffered damage to their brains or spinal cords. At present, injuries to the brain and spinal cord are irreversible and life threatening.